The role of calcium ions (Ca) in nerve cell excitability was investigated in sympathetic neurons of the bullfrog using intracellular and sucrose gap recording techniques. In sodium free Ringer's solution, isotonic Ca, strontium (Sr) and barium (Ba) are each capable of supporting regenerative action potentials. The Ca-spike elicits an after-hyperpolarization (AH) that results from activation of a potassium (K) conductance. In Ba, this AH is absent and repetitive spontaneous action potentials occur. In normal Ringer's, the administration of theophylline (5mM) or the intracellular injection of citrate ions results in the generation of spontaneous rhythmic hyperpolarizations that are generated by an increased K conductance. The frequency of the hyperpolarizations is greatly reduced or abolished by Ca-free Ringer's or Ringer's containing dantrolene sodium (0.54 uM) or in which water is replaced by deuterium oxide. Membrane excitability is significantly decreased during the spontaneous hyperpolarizations. The results indicate that both extracellular and intracellular Ca appear to be involved in the regulation of membrane excitability.